Antibody therapy provides targeted therapeutic treatment in patients with various disorders, such as cancers and immunological diseases, and therefore has played an important role in biological research. Different approaches of targeted antibody therapy, such as antibody-drug conjugates (ADC) and engineered bispecific antibodies, have been explored. See, e.g., Doronian et al., Bioconjuage Chem. 19:1960-1963 (2008); Junutula et al., Nature Biotechnology 26: 925-932 (2008); and Carter, P. J. Immunol. Methods 248(1-2):7-15 (2001).
In the case of antibody-drug conjugates (i.e., immunoconjugates), cytotoxic drugs are generally linked or conjugated to antibodies for targeted local delivery of the drug moieties to tumors. Chemical modification has been widely used for conjugating drugs to antibodies either through lysine side chain amines or through cysteine sulfhydryl groups activated by reducing interchain disulfide bonds. However, these types of “residue-specific” conjugation lead to a heterogeneous mixture of conjugates having different molar ratios of drug to antibody, different and non-specific conjugation sites, different efficiency, safety, and pharmacokinetics, and different clearance of antibody-drug conjugates. See Tanaka et al, FEBS Letters 579:2092-2096 (2005). Further, inclusion bodies or incorrect disulfide bridges may also be formed in cysteine-introduced antibodies. See, e.g., Gentle et al., Bioconjugate Chem. 15:658-663 (2004). Reactive cysteine residues engineered at specific sites of antibodies (e.g., THIOMAB) for specific drug conjugation with defined stoichiometry has also been explored. See Junutula et al., Nature Biotechnology, 26: 925-932 (2008). However, expression and conjugation of such cysteine engineered antibodies and antibody-drug conjugates are complicated processes which require lengthy reaction procedures (e.g., reductions and oxidations). See, e.g., Gomez et al., Biotechnology and Bioengineering, 105(4): 748-760 (2009). Antibody aggregates may also be generated during the process of making the cysteine engineered antibodies and the antibody-drug conjugates.
Enzymatic approaches using a transglutaminase for protein conjugation have been explored recently as an alternative to “residue-specific” conjugation of antibodies/proteins and drugs. Transglutaminases (EC2.3.2.13; protein-glutamine-gamma-glutamyltransferse; protein-glutamine:amine γ-glutamyltransferase; CAS 80146-85-6) belong to a family of enzymes that catalyze the acyl addition to a primary amine wherein the gamma-carboxamide group of peptide-bound γ-glutanyl residue is the acyl donor and the primary amine is the acyl acceptor and the amine donor. Transglutaminases have been used, for example, for the attachment of proteins to proteins. See, e.g., Tanaka et al, FEBS Letters 579:2092-2096 (2005). Enzymatic modification of antibodies using transglutaminases has also been reported. See Josten et al. J. of Immunological Methods 240:47-54 (2000); Takazawa et al., Biotechnology and Bioengineering 86(4): 399-404 (2004); and Mindt et al., Bioconjugate Chem 19:271-27 (2008). Protein conjugation or modification using transglutaminase provides the advantages of high selectivity, simplified reaction procedures, and mild reaction conditions. However, to date, because of the substrate specificity of transglutaminase, site-specific conjugation of antibodies and proteins mediated by a transglutaminase has not been clearly established. Accordingly, more efficient methods for generating a site-specific and homogenous antibody-drug conjugate, antibody conjugate, or protein conjugate using transglutaminase are needed.
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